Nuclear magnetic resonance (NMR) spectroscopy is one of the most important and widespread analytical techniques used in the characterization of glycosaminoglycans in general, and heparins and low molecular weight heparins and their derivatives in particular.
The possibility of performing both one-dimensional and two-dimensional experiments makes this technique highly sensitive for determining small variations in molecular structure, making it very advantageous for a suitable characterization of these compounds.
Glycosaminoglycans (GAGs) are linear and negatively charged polysaccharides with a mean molecular weight between 10-100 KDa (“The Structure of Glycosaminoglycans and their Interactions with Proteins”; Gandhi N S and Mancera R L. in Chem. Biol. Drug Des. (2008), 72, 455-482). There are two large groups (genera) of glycosaminoglycans: non-sulfated (such as hyaluronic acid) and sulfated (such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, and heparan sulfate). Glycosaminoglycan chains are formed by disaccharide units or disaccharides composed of an uronic acid (D-glucuronic or L-iduronic) and an amino sugar (D-galactosamine or D-glucosamine) such as the following.
Formula 1: General structure of the disaccharide unit for the different types of glycosaminoglycans.
Heparin is a polysaccharide of the glycosaminoglycan genus of compounds, formed by uronic acid (L-iduronic or D-glucuronic acid) and D-glucosamine, linked in alternating sequence. L-iduronic acid may be 2-O-sulfated and D-glucosamine may be N-sulfated and/or 6-O-sulfated, and to a lesser extent N-acetylated or 3-O-sulfated (“Mapping and quantification of the major oligosaccharides component of heparin”, Linhardt R J, Rice K G, Kim Y S et al. in Biochem. J. (1988), 254, 781-787). The major disaccharide repeating unit corresponds to the trisulfated disaccharide, 2-O-sulfo-L-iduronic acid (1→4) 2-N-sulfo-6-O-sulfo-D-glucosamine.
The origin of this structural variability present in heparin oligosaccharide chains is found in their biosynthesis and in the mechanism regulating it. Thus, in the first stage of biosynthesis, a tetrasaccharide fragment formed by glucose-galactose-galactose-xylose is bound to a protein core, starting the biosynthesis of the glycoprotein chain. Next, glucuronic acid (GlcA) residues and N-acetylglucosamine (GlcNAc) residues are alternatively incorporated forming a polysaccharide chain of approximately 300 units. At the same as this chain elongation occurs, and due to the intervention of various enzymes, modifications occur therein. Thus, the action of N-deacetylase/N-sulfotransferase enzymes produce the N-deacetylation and N-sulfation of the GlcNAc units, turning them into N-sulfoglucosamine (GlcNS). A C5 epimerase catalyzes the transformation of certain units of GlcA into iduronic acid (IdoA), followed by a 2-O-sulfation due to action of a 2-O-sulfotransferase. Next, a 6-O-sulfotransferase, transfers a 6-O-sulfo group to GlcNS and GlcNAc units. Finally, a 3-O-sulfotransferase acts on certain N-sulfo-6-O-sulfoglucosamine (GlcNS6S) units generating N-sulfo-3,6-di-O-sulfoglucosamine (GlcNS3S6S) residues.
The apparently random and incomplete nature of the initial N-deacetylation is what is mainly responsible for the introduction of the structural heterogeneity in heparin in the first phase of its biosynthesis. Structural variability with regard to the degree and positions of sulfation is the result of the incomplete nature of modifications made by the biosynthetic enzymes that lead to the production of heparin sodium molecules with a variable disaccharide substitution pattern. Currently, no prior art NMR method nor corresponding composition for the highly accurate quantification of individual saccharides in GAG's and heparin molecules exist.
Heparin is preferably used as sodium salt, but it can also be used as a salt of other alkaline or alkaline-earth metals and is mainly used as antithrombotic and anticoagulant medicine (“Anticoagulant therapy for major arterial and venous thromboembolism”, Tran HAM, Ginsberg J S in Basic principles and clinical practice (Colman R W, Marder V J, Clowes A W, George J N, Goldhaber S Z (Ed). Lippincott Williams and Wilkins; 2006:1673-1688)).
Heparins can be classified depending on their molecular weight: unfractionated heparin (UFH), Low Molecular Weight Heparin (LMWH) with a mean molecular weight lower than 8000 Da and Ultra Low Molecular Weight Heparin (ULMWH) with a mean molecular weight lower than 3000 Da (“Chemoenzymatic synthesis of homogenous ultra low molecular weight heparins”, Xu Y. et al. in Science (2011), 334, 498-501). LMWH and ULMWH come from depolymerization of the original molecule of UFH, and its manufacturing process may introduce certain process-related characteristics in the molecule's structure. Thus, the resulting molecule's structure derives on the one hand from the structure of the heparin used as starting material and on the other hand from the characteristic residues generated during preparation and characteristic manufacturing method used.
The manufacturing process of enoxaparin sodium (β-elimination by alkaline treatment on benzyl ester of heparin in aqueous medium) and bemiparin sodium (β-elimination by alkaline treatment in non-aqueous medium) generates as the majority species at the ends 4,5-unsaturated-2-O-sulfo-uronic acid (ΔU2S), at the non-reducing end, and 2-N-sulfo-6-O-sulfoglucosamine, at the reducing end of the molecule. Additionally, the non-reducing end may have saccharides such as 4,5-unsaturated-2-O-uronic acid (ΔU). At the reducing end of the aforementioned residue, it is possible to find 2-N-sulfo-6-O-sulfomannosamine (the alkaline treatment catalyzes the epimerization in C2), in addition to another two species of 1,6-anhydro derivatives: 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A) and 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M).
Formula 2: Structures present at the reducing and non-reducing end in enoxaparin and bemiparin sodium.

Residues are also generated in other low molecular weight heparins according to their manufacturing process. For example, tinzaparin sodium, which is obtained by a method of β-elimination by treatment with heparinases, has at its non-reducing end 4,5-unsaturated-2-O-sulfouronic acid (ΔU2S).
Formula 3: Structures present at the reducing and non-reducing end in tinzaparin sodium.
Dalteparin sodium is obtained by treatment with nitrous acid which generates a 2,5-anhydro-mannitol residue at the reducing end of the molecule.
Formula 4: Structures present at the reducing and non-reducing end in dalteparin sodium.
NMR spectroscopy allows for identification of the saccharide residues typically present in heparin and low molecular weight heparin, such as those formed during respective manufacturing processes.
One of the advantages associated with the use of NMR for structural characterization is that, for its analysis, the samples do not require previous derivatization or chromatographic fractionation. In other words, the sample can be directly analysed by NMR, without the need for intermediate treatments.
NMR spectroscopy is used to determine the sequence of monosaccharide residues present in these compounds and unequivocally determines the N-acetylation and N- and O-sulfation points throughout the oligosaccharide chain. Additionally, this technique allows specifically determining the orientation of the anomeric bonds and distinguishing between the iduronic acid of glucuronic acid epimers. (“Advancing Analytical Methods for Characterization of Anionic Carbohydrate Biopolymers”, Langeslay D. J. PhD Thesis UC Riverside 2013). However, given the high degree of microheterogeneity and polydispersity of these compounds, the complete characterization of heparins and low molecular weight heparins is currently still a challenge.
NMR can also be used to obtain information on those structural residues associated with the production process of heparins and of low molecular weight heparins, such as the state of epimerization of uronic acids (iduronic acid vs. glucuronic acid), ratio of sulfated and nonsulfated 4,5-uronate residues at the non-reducing end (for low molecular weight heparins produced by a β-elimination method or treatment with heparinases).
Likewise, NMR can be used as a screening technique to determine impurities present in glycosaminoglycans (“Analysis and characterization of heparin impurities”, Beni S. et al. in Anal. Bioanal. Chem. (2011), 399, 527-539).
Various NMR methods and experiments have been disclosed for the structural characterization of glycosaminoglycans in general, and heparins and low molecular weight heparins in particular. Thus, for example, 13C-NMR spectroscopy has been used to determine the degree of sulfation in heparin sodium of different animal origin (“Characterization of Sulfation Patterns of Beef and Pig Mucosal Heparins by Nuclear Magnetic Resonance”, Casu B. et al. in Arzneim.-Forsch./Drug Res. (1996), 46, 472-477).
1H-NMR spectroscopy has been the most widely used technique for the study of these compounds, since hydrogen is an abundant nucleus with a high gyromagnetic ratio. The region between 1.8-2.1 ppm comprises the signals corresponding to the N-acetyl groups or methyl groups of the reducing ends which may be synthetically included. The region between 2.8-4.6 ppm comprises the majority of the saccharide ring signals and has a high degree of overlapping between them, which makes it difficult to extract structural information directly from this area.
Two-dimensional experiments (2D NMR) allow the shortcomings of one-dimensional experiments to be overcome, i.e. shortcomings such as the overlapping of signals. Two-dimensional spectra have two frequency dimensions and another signal intensity which allows them to become a powerful tool for assigning oligosaccharide structures derived from heparin (“Characterization of currently marketed heparin products: composition analysis by 2D-NMR”, Keire D. A. et al. in Anal. Methods (2013), 5, 2984-2994).
TOCSY (TOtal Correlation SpectroscopY) spectroscopy can be used for the structural analysis of oligosaccharides, since the information obtained in that type of analysis allows the correlation of nuclei found in the same spin system, in this case all the protons within the same monosaccharide.
Another two-dimensional experiment of particular importance for the structural characterization of this type of compounds is 1H-13C HSQC (Heteronuclear Single-Quantum Correlation), which correlates 1H proton chemical shifts with chemical shifts of 13C and permits assignment of the primary structures of oligosaccharides derived from GAGs (glycosaminoglycans) and the monosaccharide composition (“Structural elucidation of the tetrasaccharide pool in enoxaparin sodium”, Ozug J. et al. in Anal. Bioanal. Chem. (2002), 403, 2733-2744; “Structural features of low molecular weight heparins affecting their affinity to antithrombin”, Bisio A. et al. in Thromb. Hemost. (2009), 102, 865-873).
The increase in spectral dispersion achieved with this two-dimensional technique allows the quantification of the integrals of the signals which are superimposed in the corresponding one-dimensional spectra (“Low-molecular-weight heparins: structural differentiation by two-dimensional nuclear magnetic resonance spectroscopy”, Guerrini M. et al. Semin. Thromb. Hemost. (2007), 33, 478-487).
Nuclear magnetic resonance is a quantitative spectroscopy technique, since the intensity (amplitude) of the resonance lines is directly proportional to the number of resonant nuclei (spin). This, in principle, makes it possible to precisely determine the quantity of molecular structures.
The increase in intensity of the magnetic fields used in NMR has allowed the limits of detection to significantly be reduced. However, the absence of precise methods that consider and control both the experimental methods and the processing and evaluation of the spectra means that measurements made on identical samples in various laboratories may significantly differ (“Validation of quantitative NMR”, Malz F. and Jancke H. in Journal of Pharmaceutical and Biomedical Analysis (2005), 38, 813-823).
The complexity of the nuclear magnetic resonance spectra of glycosaminoglycans in general, and heparins and low molecular weight heparins and their derivatives in particular, has meant that to date no specific validation methods have been developed which allow quantification of its characteristic signals, and therefore, the suitable characterization and differentiation of these compounds. In other words, prior art NMR methods have not successfully achieved highly accurate quantitation of each of the individual saccharide residues present GAG's and heparins.
With regard to GAG pharmaceutical products, their approval by health authorities of biosimilars and/or generics of certain low molecular weight heparins, as is the case of Enoxaparin sodium, requires confirmation of similarity between the biosimilar/generic and a reference molecule. Similarity requires demonstration, among other aspects, of a suitable degree of structural similarity between both products. One of the basic aspects on a structural level that it is necessary to demonstrate is that the relative proportion of the monosaccharides that form their oligosaccharide chains and after statistical evaluation, fulfil biosimilarity criteria. For them, the method of the present invention is especially selective.
The present inventors have verified that, although the structural characterization by nuclear magnetic resonance has been widely used for characterization of these compounds, the art provides no quantitative analysis methods of glycosaminoglycan analysis by means of NMR. The absence of these methods prevents the suitable comparability between identical samples studied and assessed under not suitably established experimental conditions.
Thus, it is possible to find in the literature publications wherein the values of relative proportion which are provided for the different component residues of these compounds significantly differ from one another, which clearly indicates that they are inadequate methods (“Generic versions of enoxaparin available for clinical use in Brazil are similar to the original drug”, Glauser B. F., Vairo B. C., Oliveira C. P. M., Cinelli L. P., Pereira M. S. and Mourao P. A. S. in J. Thromb. Haemost. (2011), 9, 1419-1422).